Lyme disease caused by infection with the bacterium Borrelia burgdorferi has been characterized as the fastest growing zoonotic disease in North America. Accurate diagnosis is currently the greatest challenge for the clinical management of Lyme disease. Current methods for detection of Lyme disease in a clinical setting as approved by the CDC entail a two-tiered approach using a first tier enzyme immunoassay (EIA) followed by a second tier immunoblot for both IgM and IgG B. burgdorferi specific antibodies with whole cell B. burgdorferi lysates, recombinant antigens or various combinations depending on the commercial kit used. Although adequate, the approach suffers from certain drawbacks including the subjectivity of immunoblot analysis and lack of standardization of antigen source and lysate preparations. These challenges have resulted in discordant results between test strategies for detection of host antibodies based on the kit used largely due to lysate/antigen reagent variability and specificity of the test due to the subjective nature of the analysis. Other methods for detection of Lyme disease include live culture and approaches employing polymerase chain reaction (PCR). Live culture has shown limited success in a clinical setting, is time consuming and requires complex media that has have a limited commercial supply. PCR appears to be the most promising method for direct detection of spirochetes but has not been widely accepted for laboratory diagnosis due to low sensitivity in cerebral spinal fluid and blood and the potential false-positive results due to accidental laboratory contamination of samples with small quantities of target DNA.